1. Field of the Invention
The present invention relates to detection of the hybridization of DNA and other macromolecular events using scanning probe microscopy techniques, in particular, the use of harmonic cantilevers for tapping-mode atomic force microscope.
2. Related Art
Detection of hybridized DNA molecules has a central role in molecular biology and genetics. The hybridization reaction depends on the complementary matching of the sequence of single stranded DNA molecules. This allows one to use a single stranded DNA molecule with known sequence to identify the sequence of another DNA molecule. This fact has been widely used on DNA microarrays and similar technologies to determine the sequence of DNA molecules and to compare gene expression levels in different samples.
Atomic force microscopy (AFM) refers to a class of instruments and imaging methods where a sharp tip attached to the end of a flexible cantilever is scanned across a sample surface to map topographical features and various material properties. A variety of AFM is the tapping mode where the flexible cantilever is vibrated at one of its resonance frequencies in the vicinity of the sample. Vibration amplitude and other parameters of the cantilever motion are monitored to map the topography and material properties. The gentle interaction between the tip and the sample in tapping-mode AFM has made it the dominant operation modality of AFM.
Harmonic cantilevers and coupled torsional cantilevers for tapping-mode atomic force microscopy are specially designed cantilevers that can be used as a replacement for conventional cantilevers. When used in tapping mode, these cantilevers provide additional vibrational signals at higher frequencies (harmonics) that depend on the forces between the sample and the sharp tip of the AFM. These tip-sample forces have attractive components due to capillary forces and Van der Waals forces, and repulsive components due mainly to the stiffness of the sample. A detailed description of this technique can be found in Sahin, O., Yaralioglu, G., Grow, R., Zappe, S. F., Atalar, A., Quate, C. F. & Solgaard, O., “High resolution imaging of elastic properties using harmonic cantilevers,” Sensors and Actuators A, 114, 183-190 (2004); and Sahin, O., Atalar, A., Quate, C. F. & Solgaard, O., “Resonant harmonic response in tapping-mode atomic force microscopy,” Phys. Rev. B. 69 165416 (2004).
Kreuzer, et al., “Stretching a Macromolecule in an Atomic Force Microscope: Statistical Mechanical Analysis,” Biophys. J. 80: 2502-2524 (2001) discloses methods for calculating a force-extension curve for a given macromolecule, as theoretically modeled for a molecule of PEG.
Antognozzi, et al., “Interpretation of Contrast in Tapping Mode AFM and Shear Force Microscopy: A Study of Nafion,” Langmuir 17:349-360 (2001) discloses the use of tapping mode AFM for imaging delicate samples. The publication describes the use of shear-force microscopy (SHFM). The probe is mounted on a piezoelectric actuator, which drives the probe at a frequency close to one of its resonant modes. The tip is mounted in such a fashion that its direction of vibration is parallel to the sample surface, as the probe comes into close proximity with the sample, ˜10 nm, the amplitude of oscillation decreases due to damping from the Van der Waals interactions. Nafion is a commercially available perfluorosulfonate cation-exchange membrane (CEM) manufactured by E I du Pont de Nemours & Co. Inc. The two complementary scanning probe microscopy (SPM) techniques of AFM and TDFM were used to investigate the difference in phase contrast exhibited by two Nafion samples differing only in cation form (H+ and Cs+).
Auletta, et al., “A-Cyclodextrin Host-Guest Complexes Probed under Thermodynamic Equilibrium Thermodynamics and AFM Force Spectroscopy,” J. Am. Chem. Soc. 126:1577-1584 (Jan. 15, 2004) discloses the use of “chemical force microscopy,” which combines the resolution available through force microscopy with attractive/repulsive forces taking place between a functionalized probe tip and the sample, allowing compositional mapping of surfaces with different chemical functionalities on the basis of different adhesion properties. The publication presents a study of single host-guest (HG) complex rupture forces between β-cyclodextrin self-assembled monolayers (SAMs) and several guest molecules confined onto the surface of gold-coated AFM tips by adsorption in mixed SAMs.
Viani, et al., “Probing protein-protein interactions in real time,” Nature Struct. Biol. 7:644-647 (2000) discloses the use of a small cantilever AFM to observe individual protein interactions. The authors observed, in real time, individual Escherichia coli GroES proteins binding to and then subsequently dissociating from individual E. coli GroEL proteins. Height fluctuation (topography), rather than stiffness was measured.
Van Noort, et al., “High Speed Atomic Force Microscopy of Biomolecules by Image Tracking,” Biophys. J. 77:2295-2303 (1999) discloses an image-tracking procedure to zoom in on an individual DNA plasmid. A stand-alone AFM was used. Triangular Si3N4 cantilevers (Park Scientific, Sunnyvale, Calif.) with a spring constant of 0.5 N/m were used for tapping mode in liquid, at a frequency of 30 kHz, non-harmonic mode.
Willemsen, et al., “Simultaneous Height and Adhesion Imaging of Antibody-Antigen Interactions by Atomic Force Microscopy,” Biophys. J. 75:2220-2228 (1998) discloses imaging of individual ICAM-1 antigens in both tapping mode and adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography was reportedly caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it was shown that specific molecular recognition is highly correlated with topography. V-shaped cantilevers (MICROLEVERS, tip F, Park Scientific Instruments, Sunnyvale, Calif.) with a spring constant of 500 pN/nm were operated at frequencies between 18 and 24 kHz.
Further reports are: Hansma, H. G., K. A. Browne, M. Bezanilla, and T. C. Bruice, 1994. “Bending and straightening of DNA induced by the same ligand: characterization with the atomic force microscope,” Biochemistry. 33:8436-8441; Hansma, H. G., I. Revenko, K. Kim, and D. E. Laney, 1996, “Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids,” Nucleic Acids Res. 24:713-720; and Laney, D. E., R. A. Garcia, S. M. Parsons, and H. G. Hansma, 1997, “Changes in the elastic properties of cholinergic synaptic vesicles as measured by atomic force microscopy,” Biophys. J. 72:806-813.